As a result of recent work, it has become clear that many enzymes can function as catalysts in neat organic solvents instead of water, and that when placed in this extreme and unnatural environment enzymes exhibit remarkable new properties. The overall objective of the proposed research is to explore novel synthetic opportunities afforded by enzymatic catalysis in organic solvents, particularly for the development of asymmetric and otherwise selective transformations and for their utilization in the production of biomedically relevant compounds. Our discovery during the preceding grant period that the solvent markedly affects enzyme selectivity (including enantioselectivity, chemoselectivity, and regioselectivity) will be mechanistically investigated. To this end, we will pursue kinetic, computer simulations, and structure-function relationship experiments and examine the solvent dependence of enzyme conformational dynamics which appear to play a crucial role in enzyme action in organic solvents. Protein dynamics will be studied using 2H and two-dimensional 1H NMR spectroscopies. The ultimate goal will be to elaborate a physico-chemical rationale which will explain and predict the dependence of the stereochemistry and reaction pathways of enzymatic processes in non-aqueous media, e.g., of transesterification and aminolysis reactions catalyzed by lipases and proteases, on the fundamental characteristics of the solvent. The ability to control at will enzyme selectivity by changing the solvent will provide a heretofore unavailable means of regulation of enzymatic properties ("solvent engineering"), which will be a complementary alternative to protein engineering approaches. It will also lead to profound, rational improvements in the selectivity of enzymatic conversions, such as resolution of racemates and site-specific modification of polyfunctional organic compounds.